1. Field of the Invention
Cryptosporidium parvum is a protozoan parasite that has been implicated in numerous outbreaks of diarrheal disease in the human population. This invention relates to an isolated 41 kDa protein, CP41, specific for C. parvum; recombinant CP41(rCP41) proteins: a recombinant 36 kDa protein and a recombinant 28 kDa protein, both of which are specific for C. parvum; and the nucleic acid sequences which encode these proteins. The DNA which encodes CP41 and rCP41 can be used to specifically identify C. parvum oocytes through RT-PCR. The isolated and recombinant proteins can be used as reagents to detect antibodies in the serum of infected individuals, to make monoclonal antibodies that are specific for the native 41 kDa protein which specifically identifies C. parvum and thus distinguishes C. parvum from other Cryptosporidium species, to generate hyperimmune serum or colostrum for use in enhancing the anti-cryptosporidial response of young or immunocompromised individuals, and in vaccine development, to protect individuals from Cryptosporidium infection.
2. Description of the Relevant Art
Cryptosporidium is a protozoan that can cause acute, severe, self-limited disease in immunocompetent individuals and severe chronic diarrhea in immunocompromised individuals. The young and immunosuppressed are at particularly high risk. Worldwide, there is a much higher prevalence in children than in adults (Kehl et al. 1995. J. Clin. Micro. 33 (2): 416-418. Although in most individuals the disease is self-limiting and protective immunity develops after a primary infection, cryptosporidiosis is a major cause of death in immunodeficient hosts such as persons afflicted with AIDS. Development normally takes place in the intestinal epithelium and the transmissible stage, the oocyst, is excreted in the feces. In immunocompromised patients, cryptosporidiosis is not necessarily self-limiting and sites other than the small intestine, such as the respiratory tract, stomach, liver, pancreas, gall bladder, appendix, colon, rectum, and conjunctiva of the eye, may be affected (Fayer et al. 1997. In Cryptosporidium and Cryptosporidiosis, R. Fayer, Ed., CRC Press, New York, N.Y., page 29). Cryptosporidiosis is also a major disease of dairy and beef calves in the United States. Although a number of species of Cryptosporidium have been described, only C. parvum causes disease in both humans and calves.
Over the last decade, this protozoan parasite has been implicated in numerous outbreaks of diarrheal disease in the human population. The largest outbreak reported to date was almost five years ago in Milwaukee, Wis. where greater than 400,000 people showed clinical signs of cryptosporidiosis. The source of the parasite was traced to contaminated drinking water supplied by a municipal water treatment utility. Such widespread occurrence of Cryptosporidium oocysts in raw and treated drinking water supplies throughout the USA has raised concern that low-level endemic waterborne Cryptosporidium infections may occur commonly.
Cryptosporidium is transmitted through animal contact, person-to-person contact, and contaminated food and water. The C. parvum infection is initiated by the ingestion of oocysts, the excystation of oocysts with release of sporozoites and the invasion of gut epithelial cells by sporozoites. Thereafter, the intracellular forms mature and release new daughter merozoites which reinvade the gut epithelial cells. C. parvum also has a sexual cycle. The sexual cycle of C. parvum also occurs in the gut and results in the production of sporulated oocysts, some of which may excyst before being shed. In persistent infection of an immunocompromised host, both the merozoite and the endogenously produced sporozoite may contribute to the ongoing invasion by C. parvum. Cryptosporidium spp. are resistant to standard disinfection processes and remain infectious for long periods of time in the environment at a wide range of temperatures. This resistance is imparted by the hard outer covering of the oocyst wall that surrounds the infectious stage of the parasite, i.e., sporozoites.
The detection of Cryptosporidium parvum oocysts in environmental samples usually relies on one of three different techniquesxe2x80x94vital dye staining (e.g., Modified Ziehl-Neelsen acid fast staining), direct or indirect immunofluorescence staining (IFA), or enzyme immunoassay (EIA) using Cryptosporidium-reactive antibodies. Differences in the relative sensitivities of these assays have been noted (Garcia et al. 1997. J. Clin. Micro. 35 (6): 1526-1529; Graczyk et al. 1996. Am. J. Trop. Med. Hyg. 54(3): 274-279; Ignatius et al. 1997. Euro. J. Clin. Micro. Inf. Dis. 16: 732-736; and Kehl et al. 1995. J. Clin. Microbiol. 33: 416-418). The majority of immunocompetent patients, when initially symptomatic, have large numbers of oocysts present in their stools and their condition can be confirmed with a number of procedures; however, as the acute infection resolves, the patient becomes asymptomatic and the number of oocysts dramatically decreases (Garcia et al. 1997, supra). Low numbers of oocysts makes identification of C. parvum as the causative agent difficult. The high sensitivity of anti-Cryptosporidium monoclonal antibodies (mAbs) most certainly aids detection of Cryptosporidium in fecal or environmental samples; however, their use does not ensure the specific detection of C. parvum, the only species that represents potential public health threats. Cryptosporidium oocysts shed by a variety of captive and wild homoiothermal and poikilothermal animals contaminate the surface water and water supply. In the absence of C. parvum-specific mAbs, such oocysts can be misinterpreted as C. parvum oocysts potentially pathogenic for humans based on their identification as Cryptosporidium oocysts by crossreactive antibodies, i.e., antibodies that react with more Cryptosporidium species than C. parvum (Graczyk et al. 1996. Am. J. Trop. Med. Hyg. 54(3): 274-279). Similarly, diarrhea in patients may be inaccurately diagnosed as resulting from C. parvum under circumstances where an organism other than C. parvum is the causative agent and the patient carried Cryptosporidium oocysts (not C. parvum) from contacts not related to the diarrheal disease, i.e., environmental contacts. This problem is of particular concern for water treatment utilities that must monitor the efficiency of filtration processes and the contamination level of treated water destined for human consumption. None of the available immunoassays can differentiate C. parvum from other species of Cryptosporidium that are not infectious for mammals. The inability to sensitively detect and differentiate Cryptosporidium at the level of species or subspecies (strain) is a recognized constraint on our understanding of the natural history, epidemiology, and zoonotic potential of Cryptosporidium isolates and therefore makes the assessment of the public health risk posed by oocyst contamination of water or foods difficult (M. J. Arrowood. 1997. In Cryptosporidium and Cryptosporidiosis, R. Fayer, Ed., CRC Press, New York, N.Y., page 56).
Confirmatory diagnosis of cryptosporidiosis in patients is often carried out by assaying sera for recognition of specific Cryptosporidium antigens (Frost et al. 1998. Epidemiol. Infect. 121: 205-211). Several low molecular weight C. parvum oocyst antigens, such as 15 kDa, 17 kDa, and 23 kDa proteins, appear to be useful for identifying the presence of Cryptosporidium. The immunogenicity of 15, 17, and 23 kDa antigens and somewhat higher Mr antigens (e.g., 32, 47 kDa) has been observed in other mammalian species infected or immunized with C. parvum oocysts (Lorenzo et al. 1995. Vet. Parasitol. 60: 17-25; McDonald et al. 1992. Parasite Immunol. 14: 227-232; Nina et al. 1992. Infect. Immun. 60: 1509-1513; Peeters et al. 1992. Infect. Immun. 60: 2309-2316; Perryman et al. 1996. Mol. Biochem. Parisitol. 80:137-147; Reperant et al. 1994. Vet. Parasitol. 55: 1-13). However, laboratory studies have shown these immunodominant antigens and other oocyst/sporozoite proteins to be present in other Cryptosporidium species (Nina et al. 1992, supra; Tilley et al. 1990. Infect. Immun. 58: 2966-2971); therefore, their presence is not indicative of C. parvum infection. This cross-reactivity of immunodominant antigens may explain why commercial antibody-based tests cannot differentiate C. parvum from species of Cryptosporidium that are not infectious for humans.
No antibiotics or antiprotozoal drugs licensed for animal use have been approved for prophylaxis or therapy of cryptosporidiosis (Fayer et al. 1997. In Cryptosporidium and Cryptosporidiosis, R. Fayer, Ed., CRC Press, New York, N.Y., pages 20 and 30-31). Several researchers have shown, however, that in calves, mice and humans, administration of hyperimmune bovine colostrum, prepared by immunizing cows with extracts of C. parvum oocysts, can effectively confer passive immunity against cryptosporidiosis (Fayer et al. 1989. J. Parasitol. 75(1):151-153; Fayer et al. 1989. J. Parasitol. 75(3):393-397; Fayer et al. 1990. Infect. Immun. 58(9):2962-2965; Nord et al. 1990. AIDS 4:581-584; Tzipori et al. 1986. Br. J. Med. 293:1276-1277; Tzipori et al. 1987. Lancet 2:244-245; and Ungar et al. 1990. Gastroenterology 98:486-489). Duodenal infusions of hyperimmune bovine colostrum have been reported to ameliorate C. parvum infection in AIDS or other immunocompromised patients. Hyperimmune bovine colostrum prepared against oocysts contains neutralizing antibodies that recognize epitopes expressed by all life-cycle stages of Cryptosporidium.
Monoclonal antibodies and immune sera that bind to C. parvum sporozoites can neutralize the parasite and either prevent or lessen the severity of infection in animals. The characteristics of many mAbs which specifically react with Cryptosporidium have recently been reviewed; many are neutralizing (Riggs. 1997. In Cryptosporidium and Cryptosporidiosis, R. Fayer, Ed., CRC Press, New York, N.Y., Chapter 6). In some instances, the epitope recognized by the monoclonal antibodies has been found in both sporozoites and merozoites. These monoclonal antibodies have been shown to prevent or attenuate infection in studies using animals challenged with C. parvum. However, none of these mAb specifically bind exclusively to C. parvum. Thus, they cannot be used to specifically identify the presence of C. parvum in environmental or patient samples.
Riggs reviews other mAbs reactive with surface membrane antigens and/or apical complex organelles of sporozoites and merozoites; however, no neutralization data are reported (Riggs. 1997, supra). Based on the proteins bound by these mAb and the species involved, these mAb do not selectively identify C. parvum-specific proteins.
Although protection against C. parvum may be achieved by this type of immunotherapy, the development of resistance to cryptosporidiosis is dependent upon T lymphocytes and secreted lymphokines, in particular, gamma-interferon (Gardner. 1991. Am. J. Trop. Med. Hyg. 44(1):49-62; Mead et al. 1991. J. Infect. Dis. 163:1297-1304; McDonald et al. 1992. Infect. Immun. 60 (8):3325-3331; and Ungar et al. 1991. J. Immunol. 147 (3):1014-1022). The humoral response leading to production of protective antibodies specific for C. parvum may be dependent upon T cell signaling, but in persons with severe immunodeficiency T cell-mediated immunity is dysfunctional. Passive administration of hyperimmune serum or colostrum that is inhibitory for cryptosporidial parasites may be the only viable alternative for preventing or treating infection in such individuals.
Thus, there is a need for agents useful for the immunotherapy of cryptosporidiosis in both uncompromised and immunocompromised subjects, e.g. AIDS patients, which would prevent or limit the disease. There is also a need for an agent useful for detection of C. parvum in environmental samples and for the detection of ongoing C. parvum invasion, particularly in its early stages.
We have identified C. parvum-specific antigens. Isolated and recombinant C. parvum-specific proteins can be used in a number of different ways. First, the recombinant antigens can be adsorbed to the surface of microtiter plates or to immunoblotting membrane and used in an ELISA format for detection of antibodies in serum of patients exhibiting clinical signs of cryptosporidiosis. Second, the recombinant antigens can be used to prepare monoclonal antibodies (mAb) which selectively identify or which are specific for C. parvum oocysts. These mAbs can be used in ELISA and in IFA to detect the parasite in diarrhea or in water samples similar to Cryptosporidium spp.-binding mAb used in commercial diagnostic kits (e.g., Merifluor). Third, the recombinant antigens can be used to generate hyperimmune serum or hyperimmune colostrum or in vaccine development to treat or prevent cryptosporidiosis. Fourth, primers directed to C. parvum-specific regions of the DNA sequences can be produced for sensitive detection of the parasite by polymerase chain reaction (PCR), particularly RT-PCR. PCR assays have been developed in a number of laboratories, including our own, to detect less than 10 oocysts in a spiked water sample, but the previously identified primers, as well as primers generated from the nucleotide sequences of SEQ ID NO:1, amplify DNA from all species of Cryptosporidium. However, primers can be used in RT-PCR to specifically identify transcription of C. parvum-specific proteins.
We have now discovered a novel recombinant DNA clone designated rCP41 which encodes an oocyst wall protein of Cryptosporidium parvum, which may have immunodiagnostic potential for cryptosporidiosis as well as potential for use in the production of hyperimmune colostrum that may be used to confer passive immunity against the parasite. The DNA sequences may be inserted into DNA molecules such as cloning vectors or expression vectors for the transformation of cells and the production of the C. parvum-specific proteins.
In accordance with this discovery, it is an object of the invention to provide new proteins and peptides, comprising all or part of the amino acid sequences shown in SEQ ID NO:2, that are specific for C. parvum and that can bind antibodies specific for C. parvum and elicit an immune response specific for C. parvum. This invention, in addition to the above, also encompasses a method of diagnosing Cryptosporidium infection of a subject, comprising: contacting a body fluid obtained from the subject with the peptide of this invention; and detecting any selective binding of the peptide to any anti-Cryptosporidium antibodies in the body fluid.
It is also an object of the invention to provide new proteins and peptides that are specific for C. parvum and that can therefore be used to generate antibodies for identifying the presence of C. parvum in biological samples and in water. It is part of this invention to provide the genes which encode these peptides.
In particular, this invention comprises a method of diagnosing Cryptosporidium infection of a subject, comprising: contacting a body substance obtained from the subject with an anti-C. parvum antibody; and detecting any selective binding of the antibody to any antigenic C. parvum-specific peptide present in the body substance.
Further, as a public health issue, there is a need for a method to identify and enumerate the presence of Cryptosporidium parvum in water. This invention comprises a method of identifying the presence of C. parvum in water, comprising: contacting a sample from the water source, or a concentrate of a sample from the water source, with an anti-C. parvum antibody; and detecting any selective binding of the antibody to any antigenic C. parvum-specific peptide present in the water.
It is also an object of the invention to provide new proteins and peptides that are specific for C. parvum and that can therefore be used to generate monoclonal or polyclonal antibodies, hyperimmune serum, or hyperimmune colostrum for use in immuno-therapeutic methods for preventing and treating C. parvum infection. It is part of this invention to provide the genes which encode these peptides.
Another object of the invention relates to a method of inhibiting or ameliorating a Cryptosporidium infection in an individual comprising administering to an individual in need of such treatment an amount of an anti-CP41 or anti-rCP41 antibody or hyperimmune colostrum or hyperimmune sera effective to prevent or decrease the severity of cryptosporidiosis.
It is an additional object of this invention to provide new proteins and peptides, and the genes which encode them, that are effective for the immunization of animals against cryptosporidiosis.
An added object of the invention is to provide compositions and methods useful for protecting animals against cryptosporidiosis.
Another object of the invention relates to a method of protecting an individual comprising administering to an individual an amount of proteins and peptides of this invention capable of eliciting from the individual a B- or T-cell immune response effective to prevent or to decrease the severity of cryptosporidiosis.
Further, the invention can comprise fusion proteins comprising one of the peptides described above comprising one or more epitopes of rCP41 protein wherein said rCP41 protein comprises an amino acid sequence shown in SEQ ID NO:2 and wherein said protein is antigenic and effective to elicit an immune response against Cryptosporidium parvum in a host animal and a second unrelated peptide expressed by a regulatory DNA segment operably coupled to the DNA segment described above that encodes the peptide of this invention. In addition, the invention can comprise fusion proteins comprising the unrelated peptide expressed by a regulatory DNA segment operably coupled to a DNA nucleotide sequence encoding a fusion protein comprising one of the peptides described above comprising one or more epitopes of rCP41 protein wherein said rCP41 protein comprises an amino acid sequence shown in SEQ ID NO:2 and wherein said protein is antigenic and effective to elicit an immune response against Cryptosporidium parvum in a host animal operably coupled to yet another unrelated polypeptide sequence (different from the regulatory protein). It is part of this invention to provide the genes which encode these fusion proteins. Still part of this invention are fusion RNA and DNA polymers comprising the RNA or DNA of this invention and a second unrelated polyRNA or polyDNA segment.
Additionally, it is an object of the invention to provide DNA primers from the sequence described in SEQ ID NO:1. The invention further comprises a method for specifically identifying C. parvum which comprises amplifying a subject mRNA by the RT-PCR method with the use of the above-mentioned DNA primers and thus assaying the expression of the CP41 gene.
Also part of this invention is a Cryptosporidium diagnostic kit, comprising anti-Cryptosporidium-specific antibodies; and instructions for the use of the kit.
Furthermore, this invention also provides a Cryptosporidium diagnostic kit, comprising the proteins and peptides of this invention; and instructions for use of the kit.
Other objects and advantages of this invention will become readily apparent from the ensuing description.